: Lymphocytic choriomeningitis virus (LCMV) provides one of the most valuable model systems in the field of viral pathogenesis. Studies with LCMV will improve our understanding of clinically important human pathogens, including Lassa fever virus and other emerging arenaviruses that pose a real threat to human health. A newly developed reverse genetic system allows now to conduct a detailed characterization of the cis-acting signals and trans-acting factors involved in virus RNA synthesis, control of gene expression, maturation and budding. This system also provides the foundations to rescue infectious LCMV entirely from plasmids. The ability to generate predetermined mutations within the LCMV genome will contribute to the elucidation of the molecular mechanisms underlying virus-host interactions, including viral persistence and associated disease, which are the long-term goals of the proposed studies with the following specific aims: 1)Characterization of cis-acting signals involved in the regulation of LCMV will identify and molecularly dissect the viral promoters. Likewise, mutational studies will determine the role of the panhandle structure and intergenic regions in virus RNA synthesis. 2) Investigation of the molecular bases of the Z-mediated inhibition of LCMV minigenome expression. The sequence and structural requirements of Z to exert its inhibitory activity will be determined. Studies will be done to elucidate the interactions of Z with virus RNA, as well as L and NP proteins that contribute to Z's inhibitory activity. 3) Production of infectious LCMV from DNA. Cis-and trans-acting requirements and optimal experimental conditions for packaging and budding of LCMV minigenomes will be defined. Plasmid derived S and L genomic RNAs containing genetic tags will be constructed and intracellularly coexrpessed with the viral trans-acting proteins required to initiate a productive infectious cycle, thus resulting in the rescue of infectious LCMV. This will provide a new and powerful approach to the study of the arenavirus molecular biology and pathogenesis.